Analysis of a large number of complex samples at low quantity and limited sample volume requires an analytical technique with fast analysis time, high specificity, high sensitivity, and high resolution. To achieve high resolution and high sensitivity under electrospray ionization (ESI) technique, separation techniques that use narrower columns/capillaries with low flow rates, such as nano-liquid chromatography (nano-LC) and capillary electrophoresis (CE), have become wide spread. For example, in high performance liquid chromatography-mass spectrometry (HPLC-MS), 75-μm-i.d. reverse phase columns with flow rates of ˜250 mL/min (nano-LC) have become the column of choice for proteomics study. In nano-liquid chromatography-mass spectrometry (nLC-MS), as long as the flow rates in the analytical capillaries are >100 mL/min, electrical connection to the end of these capillaries for electrospray ionization is usually achieved through a “zero dead volume” union/Tee.
In conventional capillary electrophoresis mass spectrometry (CE-MS), where a 50 or 75-μm-i.d. capillary is usually used, sheath-flow interfaces that provide voltage to the capillary outlet through addition of conductive liquid are utilized. To achieve higher resolution and more sensitivity, even narrower columns with lower flow rates such as nLC-MS using column diameter of less than 75-μm, or alternatively CE-MS with narrower capillaries (<30-μm-i.d.) with flow rates in the low nL/min are gaining popularity [1]. At these low flow rates however, conventional interfacing techniques, such as attaching a nano-spray tip to the analytical capillary using a union, are no longer useful because of the dead volume that they introduce in the interface region.
Over the years, three general techniques have been developed to address the need for interfacing narrow capillaries with low flow rates to MS via ESI: sheath-flow, sheathless, and split-flow interfaces [2-11]. Sheath-flow techniques bear several disadvantages: (1) dilution of the analyte by the sheath liquid; (2) competition for available charge between the species present in the sheath-flow and the analyte in the ESI process (Gale and Smith 1993); and (3) effects on separation, solubility, or molecular conformation which vary according to sheath liquid composition (Thompson et al. 1993, Foret et al. 1994, Smith et al. 1991). Therefore, in recent years, sheathless and split-flow interfaces have become more popular for interfacing low flow rates capillaries to MS because of their higher sensitivity of detection, which results from the absence of a sheath liquid to dilute the capillary effluent.
In split-flow techniques, a small portion of the capillary flow is diverted outside of the capillary through a small hole near the capillary outlet [12]. However, when applied to capillaries with i.d. <30-μm, controlling the split ratio using mechanical tools was difficult. This disadvantage was eliminated with a porous junction design [2], in which an electrical connection to the CE capillary outlet was achieved by making a small section of the capillary near the outlet porous. After sharpening the capillary outlet tip, the porous junction was inserted into the existing ESI needle (or sheath metal tubing) filled with a conductive solution (background electrolyte-BGE). Application of high voltage to the sheath metal containing BGE causes oxidation (in positive mode) or reduction (in negative mode) of water (if aqueous solution was used as the BGE). Ion-transport through the porous junction closes the CE circuit and provides voltage for ESI. In this design it is ion and not liquid transport through the porous section that provides electrical connection to the capillary outlet.
The use of ion transport through a porous section of a capillary for closing the electrical circuit has been employed before including: (1) a nanospray tip attached to the CE capillary outlet using polysulphone microdialysis tubing [13], (2) a liquid junction through a porous segment around the entire circumference of the capillary near the outlet [14], and (3) through a porous glass joint [15]. However, the major disadvantage of attaching a nanospray tip to the capillary outlet using polysulphone microdialysis tubing is that because the capillary inner diameter is usually smaller than the wall thickness, there is a relatively large dead volume where the two capillaries are joined. The draw back of employing a liquid junction for making the electrical connection is that since the entire circumference of the capillary was etched till porous, the porous section of the capillary is very weak and requires a liquid junction to hold the two segments of the capillary (before and after the porous section) together.
Making a section of a capillary porous has also been used in CE for other purposes than CE-to-MS interfacing. For example, porous capillary at the inlet end was recently used for the on-line concentration of proteins and peptides in capillary electrophoresis in which a short length along the capillary (around the entire circumference of the capillary) was etched with HF [16]. In CE with electrochemical detection, porous CE capillaries have been used to isolate the electrochemical detector from the CE electrical field [17-21]. In our previously reported porous junction design [2], because only a very small section of the circumference of the capillary is made porous, the capillary maintains its integrity. In addition, the inner wall of the capillary remains intact and therefore, no dead volume was introduced into the capillary. Moreover, since no liquid is added to the capillary outlet, the porous junction design provided high sensitivity. However, there were two disadvantages with the porous junction design. Because mechanical tools are used to make the well to produce porous junction, fabrication of the interface on a reproducible and large scale is impractical. In addition, the capillary outlet has to be sharpened in a separate process. Furthermore, lack of an automated, robust, and reproducible method for interfacing narrow capillary with low flow rates to MS has, for example, prevented CE-MS to become a wide-spread separation technique.